Medicinal orobancheace extracts

ABSTRACT

Pharmaceutical extracts from Egyptian broomrape have proven efficacy against HCV and NAFLD. Compositions and method of making and using same are provided.

PRIOR RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.14/625,059, filed Feb. 18, 2015 and a divisional of U.S. applicationSer. No. 13/872,699, filed Apr. 29, 2013, which is acontinuation-in-part of WO/2012/143017, filed on Oct. 25, 2011, whichclaims priority to Egyptian Application 2011040596, filed on Apr. 17,2011. Each of these applications is incorporated by reference in theirentirety herein for all purposes.

FIELD OF THE DISCLOSURE

The disclosure generally relates to methods and compositions fortreating hepatitis C virus (HCV) and non-alcoholic fatty acid liverdisease (NAFLD), as well as method of making such compositions.

BACKGROUND OF THE DISCLOSURE

Orobanchaceae—the Broomrape—is a family of flowering plants of the orderLamiales, with about 90 genera and more than 2000 species, many of whichgenera were formerly included in the family Scrophulariaceae sensu lato.Together they are a monophyletic group, forming a distinct family, foundmainly in temperate Eurasia, North America, South America, parts ofAustralia, New Zealand, and tropical Africa.

The Orobanchaceae are annual herbs or perennial herbs or shrubs, and all(except Lindenbergia and Rehmannia) are parasitic on the roots of otherplants—either holoparasitic or hemiparasitic (fully or partlyparasitic). The holoparasitic species completely lack chlorophyll andtherefore cannot perform photosynthesis. They may be yellowish,brownish, purplish, or white. Their alternate leaves have been reducedto somewhat fleshy, sessile scales. The hemiparasitic species(transferred from Scrophulariaceae) are capable of photosynthesis, andmay be either facultative or obligate parasites.

Naked Broomrape was used historically by some North American tribes forexternally treating ulcers and cancerous growths, and internally fortreating some bowel disorders. California Broomrape or cancer root isused externally to treat skin infections caused by Streptococcus, as apoultice or wash for wounds, ulcers and herpes lesions. Ancient Chineseherbalists have also used Broomrape varieties for various ailments. Forexample, Orobanche cumana is said to have the effects of antifatigue,facilitating immune function and an andrin-like action.

However, this family is mostly famous in a negative way. It is widelyscourged for the tremendous damage to crops caused by some species inthe genera Orobanche and Striga.

To date, broomrape extracts have never been used to treat HCV or NAFLD.

SUMMARY OF THE DISCLOSURE

Pharmaceutical compounds have been extracted from a member of the familyOrobancheace and have been proven to have an effect against HCV infectedcells and induced nonalcoholic fatty liver in laboratory models.

The plant is identified as a member of the family Orobancheace, speciesaegyptiaca. Orobanche aegyptiaca is a species of Broomrape, commonlyknown as Egyptian Broomrape, and is a obligate parasite of a variety ofvegetable and some ornamental species. Shown in FIG. 1, the stem isabout 15-50 cm, usually branched, yellowish, up to 6-8 mm thick in itsmidsection, with slight thickening at base, and with someovoid-lanceolate scales up to 15 mm length. One plant produces over 1million seeds every year. The seeds maintain their germinating capacityin soil for a long time under favorable conditions. The parasite preferscultivated (rarely wild-growing) plants of more than 90 species indifferent families of flower plants (except monocotyledons).

This species is found in the lower Volga, Crimea, the Caucasus, CentralAsia (in the former USSR), the eastern Mediterranean, Central Asia andAsia Minor.

The parasite occurs in fields, in watermelon plantations, inwild-growing plants, usually near roads and along field edges, but israrely seen on stony slopes. It is the most dangerous parasite of allBroomrape species, parasitizing melons, cucumbers, watermelons,pumpkins, tomatoes, red pepper, eggplants, potatoes, maize, tobacco,groundnut, and, though more rarely, sunflowers, cabbage, mustard,radish, carrots, and sesame.

Generally, the extraction method is as follows:

1) Dried powdered whole plant is mixed with 10-20 fold of sterileextraction solution, which can be water or any aqueous buffer solution,plus or minus detergents, and the like.

2) The mixture is heated to a boil under 1-2 kilo Pascal pressure for10-20 minutes.

3) The liquid extract is collected, filtered if necessary, and thenlyophilized.

4) Different concentrations of the lyophilized extract were then testedagainst lab cultured HCV replication and progression using publishedmethodology.

Other extraction methods can be employed, as suitable for water solublecomponents. For example, such methods include supercritical CO₂extraction, steam extraction, aqueous two-phase systems, and the like.If desired, the dried plant can first be lysed before extract, e.g., bygrinding, organic lysis, enzymatic lysis of plant cell walls, or can bephysically lysed by freezing and thawing before extraction, and thelike. Defatting procedures can also precede aqueous extraction, forexample, the material can be defatted with petroleum ether ormethanol/chloroform, and the like.

Furthermore, crude aqueous extracts can be further purified, forexample, by size exclusion chromatography, ion exchange chromatography,HPLC, precipitation, crystallization, and the like.

The crude extract has proven its efficacy in an induced nonalcoholicfatty liver in animal model, as well as ex vivo activity against HCV inPCMB cells.

The active ingredient or ingredients of the aqueous extract of the plantmaterial can combined with other active ingredients, but preferably areused alone. The active ingredient or ingredients of the aqueous extractof plant material can be used as is, or can be formulated with knownpharmaceutically acceptable carriers, diluents and/or excipients

For example, gelatin capsules containing dried aqueous extract can beproduced containing a suitable dose of the active ingredient(s).Optionally, packets containing the dried extract can be provided formixture with e.g., hot fluids, to be taken orally. The extract can alsobe formulated with solid carriers for pressing into pill forms,especially with delayed release excipients for formulating once a daypill forms.

It may also be possible to prepare forms of the active ingredientssuitable for non-oral routes of administration, such as inhalational,buccal, sublingual, nasal, suppository or parenteral dosage forms.

We also used TLC and HPLC to further purify the active ingredient(s) tobe further studied and to determine their efficacy, although this workis ongoing. The different compounds and their structure both are beinganalyzed and determined. Meanwhile, the primary compounds beingidentified are glycosylated compounds of natural origin.

This aqueous extract has been fractioned by HPLC producing 10 differentfractions that are tested for their anti-HCV efficacy. For thesteatohepatitis (also known as fatty liver disease) the totallyophilized extract was used for the treatment. It is proposed that theextract contains compounds that bind with the virus and preventing itscellular entry and contains a fatty acid or protein that may engulf andcoat the genomic material (positive strand RNA) of the virus preventingits antisense synthesis.

Both tests for the anti-steatohepatitis and anti-HCV efficacy wereexecuted and proven to be efficacious. HPLC fractions are being retestedcurrently for the treatment of HCV.

In more detail, the invention may comprise one or more of the following:A composition comprising an aqueous extract of Orobanche aegyptiacatogether with a pharmaceutically acceptable carrier; a method oftreating hepatitis C virus (HCV), comprising administering an effectiveamount of the composition of claim 1 to a patient with HCV; a method oftreating non-alcoholic fatty acid liver disease (NAFLD), comprisingadministering an effective amount of the composition described to apatient with NAFLD; a method of preparing the pharmaceutical compositiondescribed, said method comprising comminuting a Orobanche aegyptiaca,extracting a water soluble component from said comminuted Orobancheaegyptiaca and adding a pharmaceutically acceptable carrier to saidwater soluble component; a method of preparing the pharmaceuticalcomposition herein, said method comprising comminuting a Orobancheaegyptiaca, extracting a water soluble component from said comminutedwhole plant, lyophilizing said water soluble component; and adding apharmaceutically acceptable carrier to said lyophilized water solublecomponent.

The term “plant material,” as used herein, refers to any part or partsof a plant taken either individually or in a group. Examples include,but are not limited to, leaves, flowers, roots, seeds, stems, rhizomes,tubers, and other parts of a plant, including those plants describedherein as potential plant species of the invention. Preferred plantmaterial herein includes the stem.

The term “aqueous extract,” as used herein, refers to a compositionprepared by contacting plant material with an aqueous solvent followingstandard procedures such as those described herein. The term encompassescrude extracts, prepared by a simple aqueous extraction, as well ascrude extracts that have been subjected to one or more separation and/orpurification steps, including substantially purified and purified activeingredient(s) and concentrates or fractions derived from a crude extractby subjecting the crude extract to one or more additional extraction,concentration, fractionation, filtration, condensation, distillation orother purification step. The plant extract may be in liquid form, suchas a solution, concentrate or distillate, semiliquid form, such as a gelor paste, or it may be in solid form, such as in granulate or powderform.

The term “active ingredient” includes one or more active ingredients(e.g., compounds having pharmaceutically efficacy against at least HCVand NAFALD, and possibly other diseases) isolatable from at least theEgyptian broomrape, and potentially from other broomrape species or evenother plant families. It includes both synthetic (chemically made) andnatural (derived from plants) forms of the active ingredient.

The term “isolated,” when used in reference to a compound or compoundshaving pharmaceutical activity, refers to a form of the activeingredient that is relatively free of proteins, nucleic acids, lipids,cell wall, carbohydrates or other materials with which it is naturallyassociated in a live plant.

The term “substantially purified,” when used in reference to an activeingredient, refers to a form of the active ingredient that is at least75% pure when analyzed by HPLC.

The term “purified,” when used in reference to an active ingredientrefers to a form of the compound(s) that is at least 90% pure, andpreferably is at least 95, 98 or 99% pure when analyzed by HPLC.

The terms “therapy,” and “treatment,” as used interchangeably herein,refer to an intervention performed with the intention of improving arecipient's medical status. The improvement can be subjective orobjective and is related to the amelioration of the symptoms associatedwith, preventing the development of, or altering the pathology of adisease, disorder or condition being treated. Thus, the terms therapyand treatment are used in the broadest sense, and include the prevention(prophylaxis), moderation, reduction, and curing of a disease, disorderor condition at various stages. Prevention of deterioration of arecipient's status (i.e. stabilization of the disease, disorder orcondition) is also encompassed by the terms. Those in need oftherapy/treatment include those already having the disease, disorder orcondition as well as those prone to, or at risk of developing, thedisease, disorder or condition and those in whom the disease, disorderor condition is to be prevented.

The term “subject” or “patient,” as used herein, refers to an animal inneed of treatment. The term “animal,” as used herein, refers to bothhuman and non-human animals, including, but not limited to, mammals,birds and fish.

The term “nutraceutical,” as used herein, refers to a food or dietarysupplement that protects or promotes health and/or provides a benefit toa subject, which can affect the long term health of the subject. Theterm “phytoceutical,” as used herein, refers to a plant-comprisingcomposition having therapeutic properties.

The use of the word “a” or “an” when used in conjunction with the term“comprising” in the claims or the specification means one or more thanone, unless the context dictates otherwise. The term “about” means thestated value plus or minus the margin of error of measurement or plus orminus 10% if no method of measurement is indicated. The use of the term“or” in the claims is used to mean “and/or” unless explicitly indicatedto refer to alternatives only or if the alternatives are mutuallyexclusive.

The terms “comprise”, “have”, “include” and “contain” (and theirvariants) are open-ended linking verbs and allow the addition of otherelements when used in a claim. The phrase “consisting of” is closed, andexcludes all additional elements. The phrase “consisting essentially of”excludes additional material elements, but allows the inclusions ofnon-material elements that do not substantially change the nature of theinvention.

ABBREVIATION TERM bw Body weight cDNA complimentary DNA DNADeoxyribonucleic acid dNTPs Deoxynucelotide triphosphates FBS Fetalbovine serum HCV Hepatitis C virus PBMC Peripheral blood mononuclearcell PCR Polymerase chain reaction RNA Ribonucleic acid RPMI mediumRoswell Park Memorial Institute medium RT-PCR Real Time PCR

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an illustration of the Egyptian broomrape (Prior Art).

DETAILED DESCRIPTION

The disclosure provides novel pharmaceutical extracts from broomrape andnovel uses for same.

The present invention is exemplified with respect to Orobancheaegyptiaca. However, this species is exemplary only, and the inventioncan be broadly applied to other species of broomrape with similar activeingredients. Other suitable species may include Thistle Broomrape(Orobanche reticulata), Nodding Broomrape (Orobanche cernua) andCalceolaria (Calceolaria serrata).

The following examples are intended to be illustrative only, and notunduly limit the scope of the appended claims.

Wild Egyptian Broomrape Eu2010-12

The preferred species is Orobanche aegyptiaca. Commonly known as“Egyptian Broomrape”, it is an obligate holoparasite from the familyOrobanchaceae with a complex life cycle. This parasite is most common inthe Middle East and has a wide host range including many economicallyimportant crops.

When the seeds start to germinate, it puts out a root-like growth thatattaches to the roots of nearby hosts. When a successful connection hasbeen made with the host, the parasite can grow rapidly using water andnutrients taken from the host, causing yellowing of leaves, stunting andwilting of the host plant. After emergence the parasite will grow untilit flowers, produces seed and dies. The complete cycle takes 10 to 15weeks.

Orobanche aegyptiaca is an unconventional edible plant (similar toasparagus) that belongs to the local flora in Egypt and is readilyconsumed by many people. Thus, it appears to be relatively safe toingest.

Plant Eu2010-12 was obtained by collection of several co-locatedspecimens from Alexandria farm areas that has typical specimens of thisspecies.

Aqueous Extracts

The fresh plant was collected, cleaned from other herbs and washed infresh water, then sundried. Finally the dried plant was collected andstored in a dry dark place at room temperature. Dried plant was powderedby grinding and stored in dark and cold conditions before extraction.

A crude water extract was prepared by weighing 10-20 grams of the driedand ground plant in 100-150 mls of distilled water in a covered glassbottle in the autoclave at 121-125° C. for 20-30 minutes under pressure.The extract was then filtered, followed by freeze drying. Thelyophilized extract was then sent to be fractioned by HPLC and the wholelyophilized powder was used to test for its safety and efficacy.

Toxocology and Safety Study

The herb used in this study was provided as 5 g powder sachets, by Prof.Dr. Mohammed Fakhr El-Din El-Sawy sponsored by European EgyptianPharmaceutical Industries (EEPI), Alexandria, Egypt. This powder wasprepared as described above, from the same species of broomrape. A stocksolution from the dried plant was freshly prepared, by boiling 5 g fromthe dried plant at 100 ml distilled water for 2 minutes and filtered(equivalent to 80 ml), i.e. 5 g drug present in 80 ml filtrate.

Animal Methodology

For acute and chronic studies, the filtrate was given to the mice, rats,and rabbits by intra-gastric tubes and dogs by addition to its drinkingwater at these levels 0, 1.5 and 3 g/kg body weight. To estimate thedose level 1.5 g/kg body weight, each animal having a 100 g body weightreceived 2.4 ml from this filtrate plus 2.4 ml distilled water. While,to estimate the dose level at 3 g/kg each animal having a 100 g bodyweight received 4.8 ml from this filtrate. Control groups received only4.8 ml distilled water.

These amounts (4.8 ml) for each animal divided equally and given twicedaily (2.4 ml each time).

For carcinogenicity studies, the tested drug powder was given to theanimals by its addition to their diets at different concentrationsdescribed in the methodology below.

Acute Toxicity Studies

This plant is an edible plant where people eat the stems and leaves asvegetable like asparagus, although it is not a commonly used vegetable.Therefore, it was acceptable to use it in a healthy volunteers pilotstudy. Also, it had been used to treat urinary tract infection intraditional folk medicine. The dose administered to the animals to testfor the LD 50 (equivalent to 5 gm/kg body weight) was more than 30 timesthe recommended therapeutic human dose.

Acute toxicity studies were carried out in mice and rats. Single dosesof the drug and the water as the vehicle at the same volumes were givenorally by stomach tube to adult albino Swiss mice or rats. Clinicalsigns, symptoms and mortality were recorded during a 14-day observationperiod. The LD50 values were tested.

Doses were determined based on logarithmic doses as follows: Thetherapeutic dose suggested by the PI based on pilot studies on healthyvolunteers was 5 gm in 100 ml boiled water twice daily, and the averagehuman body weight is 70 kg; using the stock solution at the level of 5gm/100 ml this dose is equal to 0.14 gm/kg body weight.

Acute Toxicity Study Results:

Rats and mice could tolerate the extract equivalent to 5 g/kg with nomortalities.

Sub Chronic and Chronic Toxicity Studies

These studies were carried out in rats and dogs. The experimental subchronic dose used was 3 gm/kg body weight.

Rats:

Three groups each of 10 mature male albino rats (160-185 g) were used.The drug was given by stomach tube twice daily for a period of 3 monthsat two dose levels 1.5 and experimental sub chronic dose (the drug wasgiven as a water extract, but the dose is calculated as g/kg bodyweight).

Meanwhile, rats of the control group were given only the water as thevehicle. The rats were fed with standard feed and provided with water adlibitum. During the experiment all animals were observed daily forgeneral conditions, and behavior. Body weight and food consumption inrats were recorded at week intervals.

After 2 weeks, 1 month and 3 months from drug administration,hematological (RBCS, WBCS, PCV, HB, phagocytosis and phagocytic index)and serum analysis for liver and renal functions [total protein,albumin, globulin, alanine aminotransferase (ALT), aspartateaminotransferase (AST), gamma glutamyl tansferase (GGT), alkalinephosphatase (ALP), urea, creatinine, glucose, triglycerides, totallipids and total cholesterol and alphafetoprotein] using commercialkits.

Kits for all biochemical studies were obtained from BIOSYSTEMS® S.A.Costa Brava 30, Barcelona (Spain), while alpha-fetoprotein kit wasobtained from ACON LABORATORIES®, INC. 4-108 Sorrento Valley BoulevardSan Diego, Calif. 92121 USA.

At the end of the experiment (after 3 months), animals were weighed andanesthetized with ether for blood collection. Gross pathologic changes,weights of several organs and histological findings were recorded.

Dogs:

Two groups each of 3 mature mongrel male dogs (7-11 kg) were used. Thedrug was added to the drinking water at the level of experimental subchronic dose. Meanwhile, dogs of the control group were given only thevehicle. After 3 months from drug administration hematological and serumanalysis were done as in rats.

Sub Chronic and Chronic Toxicity Study Results:

Daily administration of Eu2010-12 by gavage twice daily at doses of 1.5or experimental sub chronic dose in rats, and its addition to thedrinking water of dogs 3 g/kg for 3 months revealed no significantchanges on the body weights, heart rates, and other physiologicalparameters and revealed no histological alterations in different organs.The following results were obtained:

1: Eu2010-12 did not induce any significant adverse changes in bloodhematological parameters in rats and dogs (Tables 1, 2, 14).Nevertheless, the phagocytic activity at all periods after drugadministration was significantly increased (Table 7).

2: Eu2010-12 induced a significant increase in total proteins andglobulins in rats (Table 3), yet it did not exceed the normal referencerange in all animals.

3: Eu2010-12 did not induce any abnormal liver and kidney functionalchanges as demonstrated by serum biochemical analysis in rats and dogs.Interestingly, Eu2010-12 induced a significant decrease in ALP, urea &creatinine in rats (Tables 3, 4, 5, 13, 16, 17). This suggests that thisextract enhance the kidney function of the treated animal, consistentwith its use for the treatment of UTIs in traditional folk medicine.

4: Although no significant change in blood glucose level was observed inanimals receiving Eu2010-12 at the level of 1.5 gm/kg, significantdecrease in blood glucose level occurred in animals receiving 3 gm/kg(Table 2).

5: Eu2010-12 induced a significant decrease in cholesterol,triglycerides and total lipids after its oral administration for 3months in rats (Tables 2, 6).

6: Light Microscopic examination of the different organs in rats andmice revealed no significant alterations as compared to the controlanimals (data not shown).

Reproductive Studies

A fertility study was carried out in 80 male and female albino rats. Thedrug was given at the dose of experimental sub chronic dose (Rfd, whichis 10% of the animal life span that is 90 days) twice daily to males andfemales, for respectively 35 and 14 days prior to mating. Dosed malesand females were mated with non-dosed counterparts.

Dosed female rats were further treated throughout the gestation period.Control rats received the vehicle only. On day 20 of pregnancy thefemale rats were sacrificed and fetuses were delivered by caesareansection for further examination.

Another study tested embryotoxicity and teratogenicity. This study wascarried out in female animals (20 rats, 40 mice, and 16 New Zealandrabbits). Animal groups were divided equally receiving the tested drugat doses of 0 and experimental sub chronic dose respectively twicedaily.

Rats:

Two groups of 10 female rats each received the tested drug at doses of 0and experimental sub chronic dose twice daily from day 6 to day 15 ofpregnancy. Rats were sacrificed on day 20 of pregnancy. Fetuses weredelivered by caesarean section. Fetal skeleton and visceral organs wereexamined.

Mice:

Two groups of 20 female mice each, received the tested drug at doses of0 and experimental sub chronic dose twice daily from day 6 to day 15 ofpregnancy. Mice were sacrificed on day 18 of pregnancy. Fetuses weredelivered by caesarean section. Fetal skeleton and visceral organs wereexamined.

Rabbits:

Two groups of 8 female New Zealand rabbits each received the tested drugat doses of 0 and experimental sub chronic dose twice daily from day 7to day 19 of pregnancy. Rabbits were sacrificed on day 29 of pregnancy.Fetuses were delivered by caesarean section. Fetal skeleton and visceralorgans were examined.

Peri- and post-natal studies were also performed. In the peri- andpostnatal study, 10 pregnant rats received the drug at the doses of 0and the experimental sub chronic dose twice daily. Dosing was startedfrom day 16 of gestation and continued throughout the 3-week-lactationperiod. Ten other pregnant rats were used as controls. Observations onthe offspring were made at birth and at day 4, 14 and 21 days afterbirth.

Eu2010-12 did not affect fertility. Dosed males showed comparable datawith the controls when dosed at experimental sub chronic dose (Table 8).Eu2010-12 did not cause any embryotoxic or teratogenic effect (Tables 8,9, 10, 11). Likewise, it did not cause any deleterious effects on thedosed females and their offspring. Litter size, survival rate and weightgain were comparable between groups (Table 9).

These fertility studies confirmed that Eu2010-12 showed no evidence ofimpaired fertility, teratogenic or embryotoxic effects at higher dosesup to several times the recommended therapeutic dose of 10 gms daily(infusions or teas of 2 sachets daily), recommended by the PI based onprevious pilot studies on healthy volunteers that were performed on thebasis of the safety of this edible herb.

Mutagenicity and Cracinogenicity Studies

The mutagenic potential of the tested drug was evaluated by analysis ofmitotic index, chromosomal aberrations and micronucleus technique.Twenty male and female mice were equally divided into two groupsreceived the drug twice daily for 3 months at doses 0 and experimentalsub chronic dose.

Analysis of chromosomal aberrations was done on femur bone marrow. Theanimals were sacrificed 1-2 hr after injection with colchicine (4 mg/kgbody weight). Bone marrow preparation was made by extraction of bonemarrow cell with 5-7 ml of KCl (0.65%) as a hypotonic solution at 37° C.for 25 min. The cells were centrifuged for 5 min at 1000 rpm and thenfixed in methyl acetic acid (Carnoy) 3:1, then centrifuged at 1000 rpmfor 5 min. The supernatant was discarded, the pellets resuspended in 5ml of fixative and centrifugation repeated. This procedure was repeatedtwice. The cells spread into clean slides and stained with Giemsa.

The bone marrow was flushed out with 1-1.5 ml fetal calf serum andcentrifuged at 1000 rpm for 5 min. The sediment cells were then smearedonto clean slides (3 for each animal). After drying, the slides werefixed in absolute methyl alcohol for 10 minutes and stained with 10%Giemsa stain.

Animals subjected to carcinogenic studies were divided into 4 equalgroups receiving Eu2010-12 daily in the form of powder by its additionto their diets at different concentrations described in the methodology.

The carcinogenic potential of Eu2010-12 was investigated in rats anddogs by determination of alpha fetoprotein. Histological examination forcarcinogenic activity was carried in mice. For further investigating thecarcinogenic activity of the tested material in parenchymatous organs,eighty mice were equally divided into 4 groups and received the drug inthe diet daily at the following levels 0, 0.25, 0.5 and 1% w/wrespectively. Mice will be investigated after 6 and 12 months from thebeginning of the experiment.

Statistical Analysis:

The data were analyzed by ANOVA and mean values of various treatmentswere compared with control values. Results are presented as mean±S.E.and considered statistical significant if p<0.05.

Eu2010-12 did not induce any significant changes in chromosomalaberrations (fragment, deletion, ring and gap) and micronuclei afterdaily administration for 3 months in mice (Tables 11 and 12). Thecarcinogenic potential of Eu2010-12 was investigated in rats and dogsafter 3 months by qualitative determination of alpha fetoprotein intheir serum. All results in this category were negative in all animalspecies. Further studies are under investigation and will continue untilone year in mice as previously mentioned.

The mutagenicity and carcinogenicity studies confirmed that Eu2010-12had no evidence of mutagenic potentials or carcinogenic activity athigher doses up to several times the recommended therapeutic dose of 10gms/70 kg human body weight daily (infusions or teas of 2 sachetsdaily).

In summary, the extract has been shown to be safe at levels up to 1 g/kgin four species.

Animal NAFL Efficacy Studies

The present efficacy studies were conducted on 40 adult male albino ratsweighing 120-150 grams. All animals were reared under the sameenvironmental conditions regarding caging, light and temperature. Therats were fed standard rat diet and were allowed water ad libitum allthrough the experimental period (20 days).

Rats were divided into 2 main groups:

Group I (Control Group):

Included 16 rats and were subdivided into 2 equal subgroups

Subgroup Ia:

8 rats, served as normal control group, fed standard rat diet andallowed water ad libitum.

Subgroup Ib:

8 rats, served as experimental control group, fed standard rat diet andreceived Eu2010-12 in a dose of 8.5 mg/kg bw daily for 20 days.

Group II (Experimental Group):

Included 24 rats. This group was subdivided into three equal subgroups.

Subgroup IIa (Fatty Liver Induction Group):

8 rats, received a single oral induction dose of acetaminophen on day 10from the starting day in a dose of 500 mg/kg bw by oral gavage.

Subgroup IIb (Prevention Subgroup):

8 rats, received Eu2010-12 in a dose of 8.5 mg/kg bw daily for 10 daysprior to induction with a single oral dose of 500 mg/kg bw ofacetaminophen by oral gavage then sacrificed on the following day afterinduction.

Subgroup IIc (Treatment Subgroup):

8 rats, were kept at control conditions for 9 days, then received asingle oral induction dose of acetaminophen on day 10 from the startingday in a dose of 500 mg/kg bw by oral gavage. The next days the ratsreceived Eu2010-12 in a dose of 8.5 mg/kg bw daily for 10 days.

After the end of the experimental period all animals were sacrificed bydecapitation. The abdomen was opened and specimens from the livers weretaken and fixed in 10% formol saline and processed to get 10 micronparaffin sections for light microscopic examination using hematoxylinand eosin staining.

Group Ia: (Control Group):

Liver specimens of this group revealed normal hepatic parenchyma withcords of hepatocytes radiating from the central veins. The hepatic cellsappeared polyhedral in shape with well-defined boundaries andacidophilic cytoplasm. Each cell showed rounded vesicular and centrallylocated nucleus. Some binucleated cells were present. Hepatic sinusoidsappeared as narrow spaces lined by endothelial and kupffer cells. Theportal tracts showed normal duct radicals and blood vessels (data notshown).

Group Ib: (Animals Receiving 8.5 mg/kg bw):

Light microscopic examination of liver specimens of these animals showedpreservation of the hepatic architecture. Hepatocytes structure appearednearly similar to that of the control subgroup.

Group II: (Fatty Liver Induction Group): Subgroup IIa:

Liver of rats of this group showed variable degrees of hepaticaffection. Many hepatocytes appeared swollen with moderate to severecytoplasmic vacuolation and deeply stained shrunken nuclei. Thecytoplasm of some cells showed increased acidophilia. Central veins wereoccasionally congested. Sinusoids were either dilated or obliterated.Increase in the number of Kupffer cells were noticed. Therefore, theacetaminophen induced nonalcholic fatty acid liver disease.

Group II: (Fatty Liver Induction Group): Subgroup IIb (PreventiveGroup):

Rat liver of this group showed more or less normal hepatic structure andarchitecture with no cytoplasmic vacuolation of the liver cells.Therefore, the crude broomrape extract shows preventive effect in thisinduced nonalcoholic fatty liver animal model.

Group II: (Fatty Liver Induction Group): Subgroup IIc (ReceivingEu2010-12 after Induction):

Liver of rats of this subgroup depicted nearly normal hepatic lightmicroscopic appearance except for occasional dilatation of fewsinusoids. Therefore, the crude broomrape extract shows treatmentefficacy in this induced nonalcoholic fatty liver animal model.

In-Vitro Anti-HCV Screening

Peripheral blood mononuclear cells (PBMC) were prepared and theninfected with 2-5% HCV-infected serum in RPMI culture medium containing5-10% FBS. Different concentrations (10, 25, 50, 100, 250, 500 μg/ml) ofthe crude water extract of wild plant Eu2010-12 were added. Positive andnegative control cultures were included. After 48-96 h of incubation at37° C., 5% CO₂, and 95% humidity followed by total RNA extraction. Thepositive strand were detected by RT-PCR using HCV specific primers tothe 5′-untranslated region of the virus.

Afterward, HPLC fractionation was performed of the crude water extractof the wild plant Eu2010-12 resulting in 10 fractions, 100-200 μg/ml ofeach of these fractions. Both the total lyophilized extract and thefractions were examined for anti-HCV activity.

Two fractions out of the 10 HPLC fractions showed ability to preventcultured HCV replication in the in vitro test. Based on the results, itwas concluded that the total extract prevented the replication of theHCV in vitro. In addition, 2 fractions showed promising inhibition ofthe hepatitis C virus progeny.

RT-PCR of HCV RNA

Total RNA was extracted from PBMC HCV-infected cells. Briefly, culturecells were mixed with 10-15 times volume of 4M guanidiniumisothiocyanate containing 25 mM sodium citrate, 0.5% sarcosyl, 0.1Mβ-mercaptoethanol, and 100 μL sodium acetate. The lysed cells were mixedwith an equal volume of phenol, chloroform and isoamyl alcohol (25:24:1,pH 4). After vortexing of the sample, the mixture was centrifuged at12-14 K rpm for 10-15 min at 4-8° C. The aqueous layer was collected andmixed with an equal volume of isopropanol. After incubation at −20° C.overnight, RNA was precipitated by centrifugation at 12-14 K rpm for30-45 min at 4-8° C. and the precipitated RNA was washed twice with70-80% ethanol.

The complimentary DNA (cDNA) and the first PCR reaction of the nestedPCR detection system for the HCV RNA was performed in a 25-50 μL volumesingle-step reaction using the Ready-To-Go RT-PCR beads (AMERSHAMPHARMACIA BIOTECH®, Piscataway, N.J., USA), 2.5-10 μM from each of theRT downstream primer, PCR forward primer P1 and reverse primer P2. SeeEl-Hawash S A, Abdel Wahab A E, El-Demellawy M A (2006). CyanoaceticAcid Hydrazones of 3- (and 4-) Acetylpyridine and Some Derived RingSystems as Potential Anti-tumor and Anti-HCV Agents. Arch Pharm(Weinheim) 339:14-23.

The thermal cycling protocol was as follows: 30-45 min at 42° C. forcDNA synthesis followed by 5 min at 95° C. and 30-35 cycles of 30 sec-1min at 94° C., 30 sec-1 min at 55° C. and 30 sec-1 min at 72° C. Thenested PCR amplification was performed in 25-50 reaction mixturecontaining 0.15-0.2 mM from each dNTP, 2.5-10 μM from each of thereverse nested primer and the forward nested primer, 1-2 units of TaqDNA polymerase (PROMEGA®, Madison, Wis., USA) and 5-10 μL from theRT-PCR reaction product in a 1λ buffer supplied by the vendor. Afragment of 174 bp length was identified in positive samples.

Crude water extract of wild plant Eu2010-12 was capable of preventingviral replication completely at concentration range from 250-500 μg/ml,while concentrations ranging from 10-100 μg/ml showed variable degree ofviral replication prevention. At 10 μg/ml, the drug partially preventedviral replication.

In-Vitro Anti-HCV Screening

The same concentrations of the crude water extract of wild plantEu2010-12 were examined for anti-HCV activity quantitatively using sameculture system used for the qualitative test and using the ANALYTIK JENAAG HCV viral RNA extraction kit and the ROBOGEN HCV quantitative kit(BOMETERA, Germany).

Quantitative results were shown to be in agreement with previouslyqualitative results.

Drug concentration (μg/ml) HCV Viral Load (IU/ml) Positive control 1.28× 10⁹ 500 Zero 250 Zero 100 144 50 388 10 461

In summary, the extract has been shown to be safe at levels up to 1 g/kgin four species, and has been shown to have efficacy against HCV inPBMCs, and showed preventative and treatment efficacy in inducednonalcoholic fatty liver animal models.

The current studies used whole plant, but future studies will beundertaken in order to determine which part of the plant has the mostactive ingredient and at which stage of the life cycle. Peripheral bloodmononuclear cells (PBMC) can be assayed as above for these tests.Samples will be collected at various stages of the life cycle, detailscarefully noted and roots, stem, seed and the like each assayed foractivity in the PBMC HCV assay.

In addition, fractionation and active ingredient characterizationefforts will continue to identify which ingredient(s) are responsiblefor the efficacy of this herb.

The complete results are presented in the following tables.

TABLE 1 Hematological parameters of tats given (Eu 2010- 12) by gavagetiwice daily for 3 months. Parameters Hb RBCS × Drug (g/dl) PCV %10⁶/Cmm Vehicle (10 ml dist. Water/kg) Pretreatment 11.9 ± 0.32 40.0 ±0.61 6.02 ± 0.25  2^(nd) week 11.6 ± 0.40 40.2 ± 0.60 6.15 ± 0.21 1^(st) month 12.0 ± 0.68 40.4 ± 1.41 6.2 ± 0.36 3^(rd) month 11.8 ± 0.5640.8 ± 1.31 6.28 ± 0.44  1.5 g/Kg Pretreatment 11.8 ± 0.64 39.6 ± 0.726.3 ± 0.43 2^(nd) week 12.0 ± 0.56 40.0 ± 1.36 5.88 ± 0.34  1^(st) month12.2 ± 0.56 41.0 ± 2.0  5.9 ± 0.33 3^(rd) month 11.4 ± 0.57 41.6 ± 2.396.4 ± 0.25 3 g/kg Pretreatment 11.6 ± 0.38 39.6 ± 1.12 6.2 ± 0.37 2^(nd)week 12.2.064 40.1 ± 1.15 6.3 ± 0.54 1^(st) month 11.5 ± 0.52 40.0 ±1.05 5.86 ± 0.36  3^(rd) month 11.8 ± 0.88 39.2 ± 0.9  6.3 ± 0.27 Valuesare mean ± S.E. N = 6 animals

TABLE 2 Hematological and biochemical parameters of tats given (Eu2010-12) by gavage twice daily for 3 months. Parameters WBCS × GlucoseCholesterol Drug 10³/Cmm (mg/dl) (mg/dl) Vehicle (10 ml dist. Water/kg)Pretreatment 7.1 ± 0.24 66.6 ± 2.09 59.6 ± 1.29 2^(nd) week 7.2 ± 0.3165.2 ± 1.16 59.8 ± 1.59 1^(st) month 6.92 ± 0.49  66.2 ± 1.93 60.6 ±1.29 3^(rd) month 6.8 ± 0.37 63.0 ± 3.04 56.8 ± 2.78 1.5 g/KgPretreatment 6.60 ± 0.40  62.6 ± 2.36 61.4 ± 2.75 2^(nd) week 6.80 ±0.50  59.0 ± 2.12 57.4 ± 1.89 1^(st) month 7.00 ± 0.44  58.8 ± 2.25 51.8 ± 1.07* 3^(rd) month 6.6 ± 0.51 57.6.62  49.2 ± 1.07* 3 g/KgPretreatment 6.9 ± 0.64 64.4 ± 2.02 60.8 ± 1.65 2^(nd) week 7.1 ± 0.45 56.4 ± 2.06* 53.8 ± 2.54 1^(st) month 6.8 ± 0.37  57.2 ± 2.96*  51.0 ±1.52* 3^(rd) month 6.4 ± 0.51  55.6 ± 2.38*  49.6 ± 1.03* Values aremean ± S.E. N = 6 animals *Significantly different compared to control(pretreated group), P < 0.05.

TABLE 3 Serum protein profile of tats given (Eu 2010-12) by gavagetiwice daily for 3 months. Values are mean ± S.E. N = 6 animalsParameters Protein Albumin Globulin Drug (g/dl) (g/dl) (g/dl) Vehicle(10 ml dist. Water/kg) Pretreatment 5.08 ± 0.24 3.26 ± 0.27 1.82 ± 0.152^(nd) week 5.12 ± 0.12  3.2 ± 0.14 1.92 ± 0.08 1^(st) month  5.2 ± 0.223.28 ± 0.18 1.92 ± 0.08 3^(rd) month 5.28 ± 0.21  3.5 ± 0.22 1.78 ± 0.191.5 g/Kg Pretreatment 2.24 ± 0.22 3.34 ± 0.25  1.9 ± 0.07 2^(nd) week5.64 ± 0.27 3.66 ± 0.29 1.98 ± 0.02 1^(st) month 5.48 ± 0.33 3.24 ± 0.44 2.24 ± 0.24* 3^(rd) month 5.48 ± 0.21 2.88 ± 0.41  2.6 ± 0.24* 3 g/kgPretreatment 5.22 ± 0.21 3.36 ± 0.27 1.86 ± 0.15 2^(nd) week 5.52 ± 0.263.34 ± 0.23 2.18 ± 0.22 1^(st) month  6.0 ± 0.27*  3.0 ± 0.27  3.0 ±0.31* 3^(rd) month  5.7 ± 0.37*  3.0 ± 0.31  2.7 ± 0.20* *Significantlydifferent compared to control (pretreated group), P < 0.05.

TABLE 4 Effect of administration of (Eu 2010-12) tiwice daily for 3months serum AST, ALT, and ALP in rats. Values are mean ± S.E. N = 6animals Parameters Protein Albumin Globulin Drug (g/dl) (g/dl) (g/dl)Vehicle (10 ml dist. Water/kg) Pretreatment 50.2 ± 1.56 17.2 ± 0.58 81.4± 1.12 2^(nd) week 49.4 ± 2.36 16.6 ± 0.98 83.0 ± 1.52 1^(st) month 47.8± 2.71 16.6 ± 0.92 82.4 ± 1.60 3^(rd) month 46.8 ± 2.01 16.6 ± 0.46 81.2± 185  1.5 g/Kg Pretreatment 48.8 ± 2.82 19.0 ± 0.71 82.2 ± 1.96 2^(nd)week 48.0 ± 0.44 18.0 ± 0.71 80.6 ± 2.16 1^(st) month 46.4 ± 2.71 17.4 ±0.68 76.2 ± 2.44 3^(rd) month 45.0 ± 2.10 16.8 ± 1.16  73.6 ± 2.73* 3g/kg Pretreatment 47.6 ± 3.21 18.6 ± 0.51 79.8 ± 1.99 2^(nd) week 45.6 ±3.06 17.4 ± 0.60 79.2 ± 1.46 1^(st) month 44.6 ± 2.93  16.0 ± 0.71* 74.4± 2.32 3^(rd) month 44.6 ± 2.93  16.0 ± 0.71*  71.6 ± 2.91**Significantly different compared to control (pretreated group), P <0.05.

TABLE 5 Effect of administration of (Eu 2010-12) twice daily for 3months serum GGT, urea and creatinine in rats. Values are mean ± S.E. N= 6 animals Parameters GGT Urea Creatinine Drug (U/L) (mg/dl) (mg/dl)Vehicle (10 ml dist. Water/kg) Pretreatment 12.0 ± 0.71 19.2 ± 0.67 0.56± 0.05 2^(nd) week 11.8 ± 0.86 19.8 ± 1.07 0.58 ± 0.07 1^(st) month 11.2± 1.16 19.4 ± 1.75 0.56 ± 0.05 3^(rd) month 12.5 ± 1.07 18.6 ± 2.07 0.58± 0.06 1.5 g/Kg Pretreatment 12.4 ± 1.03 20.6 ± 0.93 0.60 ± 0.05 2^(nd)week 12.2 ± 0.73 19.0 ± 0.89 0.58 ± 0.03 1^(st) month 12.2 ± 0.80 19.0 ±1.23 0.48 ± 0.03 3^(rd) month 11.4 ± 0.98 17.2 ± 1.07 0.58 ± 0.03 3 g/kgPretreatment 12.8 ± 0.97 17.8 ± 1.28  0.6 ± 0.07 2^(nd) week 12.6 ± 0.8117.6 ± 2.32 0.58 ± 0.08 1^(st) month 12.2 ± 1.24 16.2 ± 1.8  0.58 ± 0.8 3^(rd) month 11.0 ± 0.98  15.6 ± 0.93*  0.40 ± 0.04* *Significantlydifferent compared to control (pretreated group), P < 0.05.

TABLE 6 Effect of administration of (Eu 2010-12) tiwice daily for 3months serum Triglycerides and Total lipids in rats. Values are mean ±S.E. N = 6 animals Parameters Triglycerides Total Drug (mg/dl) Lipids(g/l) Vehicle Pretreatment 104.4 ± 3.14 5.06 ± 0.40 2^(nd) week 100.8 ±5.13  5.1 ± 0.39 1^(st) month 100.0 ± 7.26  5.0 ± 0.47 3^(rd) month101.0 ± 6.8  5.06 ± 0.37 1.5 g/Kg Pretreatment 104.0 ± 8.74 5.02 ± 0.342^(nd) week  98.8 ± 7.12 4.88 ± 0.39 1^(st) month  95.4 ± 7.24  3.84 ±0.25* 3^(rd) month  91.6 ± 4.31  3.7 ± 0.43* 3 g/kg Pretreatment 105.0 ±7.26 4.78 ± 0.51 2^(nd) week  99.0 ± 6.98  4.5 ± 0.31 1^(st) month  94.0± 5.8*  3.74 ± 0.23* 3^(rd) month  81.0 ± 3.68*  3.54 ± 0.41**Significantly different compared to control (pretreated group), P <0.05.

TABLE 7 Effect of administration of (Eu 2010-12) by gavage twice dailyfor 3 months on phagocytic activity and phagocytic index in rats. Valuesare mean ± S.E. N = 6 animals Parameters Drug phagocytic activityphagocytic index Vehicle (10 ml dist. Water/kg) Pretreatment 22.0 ± 0.711.86 ± 0.09 2^(nd) week 23.4 ± 0.40 1.72 ± 0.03 1^(st) month 23.2 ± 0.491.82 ± 0.11 3^(rd) month 22.1 ± 0.50 1.75 ± 0.20 1.5 g/Kg Pretreatment21.0 ± 0.63 1.78 ± 0.03 2^(nd) week 24.4 ± 0.51* 1.90 ± 0.10 1^(st)month 25.0 ± 0.63* 2.04 ± 0.06 3^(rd) month 25.4 ± 0.60* 2.10 ± 0.08 3g/kg Pretreatment 22.0 ± 0.71 1.74 ± 0.05 2^(nd) week 25.8 ± 0.37* 1.94± 0.09 1^(st) month 25.6 ± 0.51* 1.86 ± 0.10 3^(rd) month 24.8 ± 0.14*1.88 ± 0.20 *Significantly different compared to control (pretreatedgroup), P < 0.05.

TABLE 8 Effect of administration of (Eu 2010-12) given by gavage on therats. Values are mean ± S.E. N = 6 animals. Dosage group (g/Kg) 0 3C-M + T-M + C-M + T-M + T-F C-F T-F C-F Adult rat data No. of treatedmales n 0 10 0 10 No. of treated Females n 10 0 10 0 No. of pregnantrats n 7 7 8 8 No. of surviving Females n 10 10 10 10 Fertility index %70 70 80 80 Litter data Average no. of implantations m 6.71 6.9 6.75 6.5No. of pregnancies No. of alive fetuses % 6.43 6.57 5.88 6.25 No. ofresorbed fetuses % 4.17 4.05 3.7 3.84 Average weight at birth (g) 5.235.08 5.09 5.32 Abnormalities % — — — — C = Control M = Male T = TreatedF = Female

TABLE 9 Effect of administration of (Eu 2010-12) by gavage on thedevelopment and viability of first generation (F1) pups during thepre-weaning period on rats. Values are mean ± S.E. N = 6 animals. Dosagegroup (g/Kg) 0 3 Mean number of pups/Litter Born  6.2 ± 0.37  6.6 ± 0.51Born alive  5.8 ± 0.30  6.2 ± 0.30 Sex ratio (meals) 49.4 ± 1.07 49.2 ±1.02 Mean pup weight (g) litter Postnatal day O(PND) males 5.14 ± 0.294.88 ± 0.33 Females 4.98 ± 0.06 4.84 ± 0.09 PND 4 males 7.92 ± 0.11Females 7.92 ± 0.11 7.74 ± 0.16 PND 4 males 7.62 ± 0.16 7.46 ± 0.31Females 15.34 ± 0.55  15.7 ± 0.43 PND 4 males 14.5 ± 0.48 14.2 ± 0.73Females 25.6 ± 0.75 26.0 ± 0.54 PND 4 males 23.8 ± 1.24 24.2 ± 1.24 No.of pups alive on PND 0 100 100

TABLE 10 Reproductive parameters in mice given (En2010-12). Dose 0 3g/Kg No. bred 20 20 No. pregnant 16 18 No. aborted 0 0 No. litterstotally resorbed 0 0 No. corpora lutea 7.0 ± 0.25  7.2 ± 0.30 No.implantation 6.37 ± 0.23  6.17 ± 0.21 Dead frtuses 0 0 Litter size 6.0 ±0.20 6.17 ± 0.22 Mean felal body weight (g) 1.1 ± 0.02 1.06 ± 0.02Abnormalities % 0 0

TABLE 11 Reproductive parameters in rabbits given (Eu2010-12) Dose 0 3g/Kg No. bred 8 8 No. pregnant 6 6 No. aborted 0 0 No. litters totallyresorbed 0 0 No. corpora lutea 7.83 ± 0.47 8.67 ± 0.49 No. implantation6.67 ± 0.42 7.67 ± 0.40 Dead frtuses 0 0 Litter size 6.33 ± 0.40  7.5 ±0.62 Mean felal body weight (g) 36.0 ± 1.29 37.67 ± 1.48  Abnormalities% 0 0

TABLE 12 Different Kinds of chromosomal aberrations in mice treated with(Eu2010-12) by gavage twice daily for 3 months. Values are mean ± S.E. N= 6 animals Parameter* Dosage group End to end g/Kg Fragment DeletionRing association 0 1.4 ± 0.2 0.6 ± 0.02 0 2.2 ± 0.3 3 1.2 ± 0.5 0.8 ±0.03 0 1.8 ± 0.3 *250 metaphases were examined per each treatment

TABLE 13 Micronucleus (MN) and mitotic index in mice treated with(Eu2010-12) by gavage twice daily for 3 months. Values are mean ± S.E. N= 6 animals [0001] Parameter Dosage Micronucleus** group MitoticMicronucleus** (Nuclear End to end g/Kg index* (Nuclear budding)fragmentation) association 0 9.2 ± 0.5 1.6 ± 0.24 0.8 ± 0.3 2.2 ± 0.3 38.4 ± 0.9 1.2 ± 0.49 0.8 ± 0.4 1.8 ± 0.3 *No of dividing cells in 500cells **Micronucleus incidence in 500 polychromatic erythrocytes.

TABLE 14 Hematological parameters of dogs given (Eu2010-12) by gavagetwice daily for 3 months. Values are mean ± S.E. N = 6 animals [0001]Parameter Hb RBCS × WBCS × Dosage group (g/dl) PCV % 10⁶ cmm 10³/cmmVehicle Pretreatment 13.0 ± 0.71 35.2 ± 1.02 5.82 ± 0.29  6.8 ± 0.51After 3 months 12.5 ± 1.08 35.6 ± 1.6  6.06 ± 0.33 6.78 ± 0.50 3 g/KgPretreatment 12.6 ± 1.21 34.8 ± 1.5   6.0 ± 0.44  6.5 ± 0.50 After 3months 12.4 ± 0.51 35.6 ± 1.84 6.16 ± 0.39 6.56 ± 0.41

TABLE 15 Serum protein profile of dogs given (Eu2010-12) for 3 months.Values are mean ± S.E. N = 6 animals Parameters Protein Albumin GlobulinDrug (g/dl) (g/dl) (g/dl) Vehicle Pretreatment  6.0 ± 0.28 3.67 ± 0.332.33 ± 0.17 After 3 months 5.87 ± 0.21 3.70 ± 0.17 2.17 ± 0.09 3 g/KgPretreatment 6.07 ± 0.19 4.23 ± 0.12 1.84 ± 0.26 After 3 months 6.23 ±0.26 4.37 ± 0.29 1.86 ± 0.24

TABLE 16 Effect of administration of (Eu2010-12) on serum AST, ALT, ALPand GGT in dogs. Values are mean ± S.E. N = 6 animals Parameter AST ALTALP GGT Drug (U/L) (U/L) (U/L) (U/L) Vehicle Pretreatment 22.4 ± 1.2119.6 ± 0.51 77.0 ± 1.82 6.4 ± 0.51 After 3 months 21.8 ± 1.36 18.8 ±1.16 78.4 ± 2.86 6.6 ± 0.68 3 g/Kg Pretreatment 21.4 ± 1.75 20.6 ± 1.2974.2 ± 2.96 6.4 ± 0.51 After 3 months 20.4 ± 137  20.2 ± 1.66 73.8 ±1.78 6.2 ± 0.58

TABLE 17 Effect of administration of (Eu2010-12) on urea, creatinine andglucose in dogs. Values are mean ± S.E. N = 6 animals Parameter UreaCreatinine Glucose Drug (mg/dl) (mg/dl) (mg/dl) Vehicle Pretreatment19.8 ± 1.07 0.35 ± 0.03 65.2 ± 1.94 After 3 months 21.0 ± 1.00 0.36 ±0.04 63.0 ± 2.40 3 g/Kg Pretreatment 20.4 ± 0.68 0.34 ± 0.03 67.0 ± 2.03After 3 months 19.6 ± 0.93 0.35 ± 0.04 64.4 ± 1.69

The following references are incorporated by reference herein in theirentireties for all purposes.

-   El-Hawash S A, Abdel Wahab A E, El-Demellawy M A (2006). Cyanoacetic    Acid Hydrazones of 3-(and 4-) Acetylpyridine and Some Derived Ring    Systems as Potential Anti-tumor and Anti-HCV Agents. Arch Pharm    (Weinheim). 339:14-23.

1) A method of preparing a pharmaceutical composition comprising anaqueous extract of Orobanche aegyptiaca together with a pharmaceuticallyacceptable carrier, said method comprising comminuting a Orobancheaegyptiaca, extracting a water soluble component from said comminutedOrobanche aegyptiaca and adding a pharmaceutically acceptable carrier tosaid water soluble component. 2) A method of preparing a pharmaceuticalcomposition comprising an aqueous extract of Orobanche aegyptiacatogether with a pharmaceutically acceptable carrier, said methodcomprising comminuting a Orobanche aegyptiaca, extracting a watersoluble component from said comminuted Orobanche aegyptiaca,lyophilizing said water soluble component; and adding a pharmaceuticallyacceptable carrier to said lyophilized water soluble component.